低氧条件下原代培养人脑胶质瘤细胞中COX-2表达与放射敏感性的关系研究

Relationship between COX-2 expression and radiosensitivity of the primary cultured human glioma cells under hypoxia condition

目的探讨低氧条件下体外培养原代人脑胶质瘤细胞中环氧合酶-2(COX-2)表达与放射敏感性的关系。方法取新鲜人脑胶质瘤组织进行原代细胞培养,待细胞稳定传代后,移入低氧环境中继续培养24 h,然后将实验细胞分为3组,未转染组常规培养,转染空载体组采用含空载体培养液培养,转染COX-2-siRNA组利用基因干扰技术,沉默人脑胶质瘤细胞内COX-2基因的表达,制备COX-2靶向siRNA转染至人脑胶质瘤细胞。转染COX-2-siRNA组确定转染成功后,3组人脑胶质瘤细胞在低氧条件下培养24 h后,均给予5 Gy的^(60)Co γ射线照射。照射后换新培养基低氧条件下继续培养24 h后,Western blot法检测各组人脑胶质瘤细胞COX-2蛋白表达情况,RT-PCR法检测各组人脑胶质瘤细胞COX-2mRNA表达情况,流式细胞仪(FCM)检测各组人脑胶质瘤细胞凋亡率。结果未转染组人脑胶质瘤细胞中COX-2蛋白及mRNA表达情况与转染空载体组比较差异均无统计学意义(P>0.05),转染COX-2-siRNA组人脑胶质瘤细胞COX-2蛋白及mRNA表达水平均明显低于未转染组(P均<0.05),细胞凋亡率明显高于未转染组(P<0.05)。结论低氧条件下,COX-2蛋白及mRNA表达增强是引起人脑胶质瘤细胞产生放疗抵抗的一个独立因素。

Objective It is to investigate the relationship between cydooxygenase-2 （COX-2） expression and radiosensitiv- ity of the primary cultured human glioma cells in vitro under hypoxia condition. Methods Fresh human glioma tissues were took to cultivate the primary cells, after the cells passage were stable, the cultivation continued for 24 hours under hypoxic conditions, then the experimental cells were divided into 3 groups. The cells were cultured routinely in the group A, and was cultured by empty carrier culture medium in the group B, the COX-2 targeting siRNA transfected into human glioma cells in group C was prepared by silent expression of COX-2 gene in human glioma cells with gene interference technique. When the transfection was successful in group C, the human brain glioma cells of 3 groups were cultured for 24 hours under hypoxic con- ditions, then were irradiated with 5Gy ^60Co gamma rays. After irradiation, the new medium continued to be cultured for 24 hours under hypoxic conditions, then the expression of COX-2 protein in human glioma cells were detected by Western and blot methods, the expressions of COX-2 mRNA in human brain glioma cells were detected by RT-PCR, and the apoptosis rate of glioma cells were detected by flow cytometry in each groups. Results There was no significant difference between group A and group B in the expression of COX-2 protein and mRNA （ all P 〉 0.05） , while the expression of COX-2 protein and mRNA in group C were significantly decreased than group A （all P 〈 0.05）, at the same time, the apoptosis rates has significantly in- creased than group A （P 〈 0.05）. Conclusion Under hypoxia condition, the increased expression of COX-2 protein and mR- NA is an independent factor that induces resistance to radiotherapy in human glioma cells.